The importance of metabolism for cancer development and progression has been increasingly recognized over the past decade. Cancer cells undergo drastic alternations of metabolism, including metabolism of glucose and lipid, and oxidative responses. These processes are highly regulated by AMPK and p53. AMPK is activated by LKB1 and AMP/ADP in response to bio-energy shortage to coordinate metabolism, cell growth, apoptosis and autophagy by phosphorylating its target proteins. Also, the tumor suppressor p53 can be activated by this type of stress to confine metabolic pathways, which favor tumor growth, and to turn on cellular pathways that lead to cell growth arrest and apoptosis by inducing a variety of genes important for these functions. The anti-tumor function of p53 is tightly regulated by its two physiological antagonists MDM2 and MDMX, which form a complex to act as a negative feedback regulator of p53. Hence, blocking this feedback regulation is the key for cellular stresses to activate p53. For instance, ribosomal stress (RS) leads to p53 activation by inducing the interaction of ribosomal proteins L11 and L5 with MDM2 and restraining the latter's activity toward p53, but defect of this interaction in MDM2C305F mice impairs this pathway. Interestingly, AMPK can act as an upstream regulator of p53 upon metabolic stress by phosphorylating MDMX at Ser342, leading to inactivation of MDMX by inducing its binding to 14-3-3, consequently activating p53. Hence, our studies suggest that MDMX may function as a crucial player under metabolic stress. Also our preliminary studies using MDM2C305F and MDMX3SA double knock-in mice showed that the double impairments of the MDM2-MDMX-p53 pathways cause significant defects of lipid and glucose metabolisms in livers upon fasting. Thus, we hypothesize that the metabolic stress-AMPK-MDMX/RS-MDM2-p53 pathways may play an important role in regulation of metabolism in both normal and cancer cells upon metabolic stress. We will test this hypothesis by addressing three specific aims. 1. To elucidate mechanisms underlying the AMPK inhibition of MDMX activity toward p53 in response to metabolic stress. A) Does S342 phosphorylation by AMPK induce nuclear localization and degradation of MDMX? B) Does S342 phosphorylation by AMPK affect the formation of the MDMX-MDM2 complex? C) Does S342 phosphorylation by AMPK induce 14-3-3-binding to S367 of MDMX? 2. To determine if the AMPK-MDMX/RS-MDM2-p53 pathways play a role in regulation of metabolism in cells and mice. A. To determine if dual defects of the MDMX-MDM2-p53 pathway impair energy metabolism under physiological and stress conditions. B. To illustrate new p53 responsive genes important for regulation of lipid and glucose metabolism by the AMPK-MDMX/RS-MDM2-p53 pathways. 3. To determine if dual defects of the AMPK-MDMX/RS-MDM2-p53 pathways accelerate tumorigenesis in two mouse tumor model systems. Completing these studies would offer molecular insight into how lipid and glucose metabolism is controlled by this pathway, and also reveal new molecule targets for future anti-cancer drug discovery.